Research on sub-cellular components is an increasingly important area of biological investigation. As part of this work, it is necessary to isolate sub-cellular organelles. Isolation includes disruption or breaking apart of cell membranes without damaging the organelles, i.e., cell nuclei, mitochondria, endoplasmic reticulum, Golgi complex, lysosomes, etc. Prior art apparatus has enabled fragments of these organelles to be isolated, however such apparatus, in general, is not able to isolate intact organelles, as observed in-vivo. For example, endoplasmic reticulum occurs as tubular and sheet-like cisternae and golgi complex appear as stacks of cisternae inside the cell. However, after cells are homogenized and fractionated, the aforesaid organelles no longer resemble their previously intact, in-vivo formations. Thus, it has been heretofore difficult to study mechanisms that maintain the structure of such intra-cellular organelles.
Many prior art homogenizers use cell suspensions obtained either by scraping the cells from a culture dish (where the cells were grown) or by centrifugation when the cells are grown in suspension. Such cells are then re-suspended in a hypotonic buffer solution. This treatment swells the cells, resulting in a weakening of the cell membranes. The membranes are then disrupted in a Dounce homogenizer which includes a glass tube-like receptacle in which the cell suspension is placed and a pestle which can fit either tightly or loosely into the receptacle. By moving the pestle up and down in the glass cylinder, cell disruption is achieved. The cell-rupturing action of a Dounce homogenizer does not result in a breakage of all cells during a single stroke of the pestle. In general, many strokes must be employed to obtain a disrupted cell population (e.g., from 25 to 50 strokes). Because of this extensive homogenization procedure, the delicate intra-cellular membranous organelles are broken and lose their structure.
A further type of homogenizer, that, at times, does enable recovery of some intact Golgi structures is the ball homogenizer developed by Balch and Rothman. The ball homogenizer has a steel ball that moves inside of a cylinder, the movement of the ball modulated by pressure applied on the sides of the cylinder using syringes. The space between the ball and the cylinder varies as a result, and cells are disrupted to a various extent by use of balls of different sizes.
A variety of other types of homogenizers are disclosed in the prior art. In U.S. Pat. No. 3,556,414 to Eberly, Jr., a compression chamber homogenizer is described wherein a mass of animal cells are compacted by a reciprocating plunger rod under pressures of at least 10,000 psi. The cell mass is then disrupted by being discharged into a relatively low pressure environment. Such homogenization action assures both complete disruption of cell membranes, as well as internal organelles. In U.S. Pat. No. 4,333,611 to Zucker et al., a cell mass is initially heated by friction to a temperature above the point of evaporation of water. The cell mass is subsequently expanded into a reduced pressure area so as to enable rupturing of the cell walls.
U.S. Pat. Nos. 3,941,317 to Kanor and 4,350,768 to Tihon et al., both describe apparatus for disaggregating tissue which involve the forcing of a cell mass through a screen-like membrane or structure. By repeated actions of the apparatus, cellular components are "disaggregated".
In U.S. Pat. No. 4,509,695 to Bessman, a tissue pulverizer is described wherein the tissue is initially frozen and then placed between a mortar and pestle that have been cooled with liquid nitrogen. The pestle is then struck with a hammer to pulverize the frozen sample. U.S. Pat. Nos. 4,307,846 to Spelsberg and 4,828,395 to Saito et al., both disclose continuous-flow type tissue homogenizers similar in construction to the Dounce homogenizer described above. In specific, such homogenizers include a generally tubular container with an inner surface defining a tissue homogenization chamber. One end of the container has an inlet for introduction of non-homogenized tissue. A pestle member is disposed within the tubular chamber and is mounted for rotative movement. Grooves about the pestle's outer surface channel force the tissue between the wall of the pestle member and the inner wall of the tubular container where homogenization occurs.
With the sole exception of the Balch and Rothman ball homogenizer, each of the above described homogenizers results not only in disruption of cell membranes but also of internal organelles. Further, the use of hypotonic solutions to assist in the cell disruption process further injures internal organelle structures.
Accordingly, it is an object of this invention to provide a cell homogenizer which leaves substantially intact, intra-cellular organelles.
It is a further object of this invention to provide a cell homogenizer which operates upon cells still attached to a surface of a cell culture dish.
It is yet another object of this invention to provide a cell homogenizer for exposing cellular domains such as apical or basolateral domain of epithelial cells grown attached to a surface of a culture dish.
It is still a further object of this invention to provide a cell homogenizer that is adapted to operate with both plant and mammalian cells.
It is still another object of this invention to provide a cell homogenizer that is adapted to operate with solid tissue and with cells grown in suspension culture.
It is another object of this invention to provide a cell homogenizer which is of inexpensive construction and may be used in the research laboratory.
It is yet another object of this invention to provide an improved cell homogenizer which operates through the application of low pressure to achieve cell wall or membrane breakage.